Project Summary/Abstract Congenital heart defects (CHDs) occur in ~1% of the births, making them the most common congenital malformations. Outflow tract (OFT) defects are found in ~30% of CHDs. To develop effective pharmacological, stem cell and regenerative therapies able to prevent CHDs in children and heal cardiovascular diseases in adults, it is essential to understanding the fundamental mechanisms directing normal heart development. Therefore, a long-term goal of our lab is to understand the mechanisms controlling cardiomyocyte (CM) production during early vertebrate development. It is critical to restrict RA levels during development, as increases in RA signaling result in conserved OFT defects in all vertebrates. The goal of this proposal is to understand the coordinated epigenetic and transcriptional mechanisms by which histone deacetylase 1 (HDAC1) and retinoic acid (RA) signaling control OFT development. Furthermore, while HDAC1 and RA receptors (RARs) are proposed to repress transcriptional activation in the absence of RA, the importance of restricting the expression of direct RA targets in heart development is not understood. Our preliminary data analyzing a novel zebrafish hdac1 mutant allele and embryos deficient for Cyp26 enzymes, which a required to degrade RA, indicate that both loss of HDAC1 and Cyp26 enzymes result in smaller OFTs due to reduced second heart field progenitor (SHFP) proliferation. Furthermore, our preliminary suggest that Ripply3, a Tbx co- factor that promotes transcriptional repression, is ectopically expressed in SHFPs of hdac1 mutant and Cyp26 deficient embryos and is sufficient to inhibit OFT development. In Aim 1, we will use tissue-specific overexpression and genetic epistasis experiments to determine if ectopic Ripply3 in SHFPs cell autonomously inhibits proliferation through interactions with Tbx1. Our preliminary results suggest that HDAC1 and RARs both associate with RA response elements (RAREs) within the ripply3 promoter. In Aim 2, we will use in vitro and vivo promoter analysis coupled with chromatin immunoprecipitation to determine the mechanisms by which HDAC1 and RAR repress ripply3 expression in SHFPs. Our preliminary results suggest misexpression of multiple factors within a HDAC1-RA controlled gene regulatory network (GRN) contribute to OFTs in hdac1 mutant and Cyp26 deficient embryos. In Aim 3, we will determine if enhanced hoxb1a expression within SHFPs contributes to OFT defects and identify additional components of the HDAC1-RA GRN the require repression within SHFPs. Altogether, these studies will dramatically improve our understanding of the epigenetic and transcriptional mechanisms that coordinate normal vertebrate OFT development. Ultimately, these studies will provide a foundation for future strategies aimed at healing and preventing CHDs in children and injured hearts in adults.